JPH01243986A - Antihuman solid cancer human monoclonal antibody, antibody-producing hybridoma and production of antibody - Google Patents

Abstract

PURPOSE:To obtain a hybridoma capable of producing an antihuman solid cancer human monoclonal antibody by fusing a heterohybridoma prepared by fusing a human lymphocyte to a mouse myelomatous cell to a lymphocyte derived from a patient suffering from solid cancer. CONSTITUTION:A human lymphocyte is fused to a mouse myelomatous cell (preferably P3U1) to provide a mouse human heterohybridoma (preferably HM-5), which is then fused to a lymphocyte (preferably a lymphocyte derived from a lymph node of a patient suffering from solid cancer) derived from the patient suffering from solid cancer to afford a (mouse.human)-human hybridoma capable of producing an antihuman solid cancer human monoclonal antibody.

Description

Detailed Description of the Invention The present invention relates to human monoclonal antibodies (hereinafter sometimes abbreviated as MoAb) that have the ability to bind to human solid tumor cells, antibody-producing hybridomas, and antibody-producing hybridomas. Regarding manufacturing methods. The human MoAb of the present invention is useful as a cancer therapeutic agent or diagnostic reagent.

Various drugs and treatment methods1 diagnostic drugs have been developed for cancer, but many of them react not only with cancer cells but also with normal tissues and cells, leading to serious side effects and misdiagnosis, so their use is prohibited. There was a limit.

In recent years, in order to overcome this drawback, therapeutic methods and diagnostic methods that utilize highly specific immune reactions have been developed. One of these is the use of anti-cancer antibodies. In the past, polyclonal anticancer antibodies were used, but the MoAb production method developed by Köhler and Milstein (K'6hlar,
G. and Milstein, C.

, Neech? -(Nature), 256.495(1
975)) has made it possible to obtain MoAb that specifically recognizes cancer-specific antigens or cancer-associated antigens on cancer cells. The use of these antibodies makes it possible to specifically bind to and eliminate cancer cells without damaging normal tissues, making it possible to develop anticancer agents with extremely high cancer tropism.

In addition, in the field of diagnostic reagents, serum, urine, or feces are highly sensitive and do not cross-react with normal components in serum, urine, or feces.
It is believed that cancer-specific antigens and cancer-related antigens in urine and feces can be detected.

However, these anti-cancer antibodies are mostly derived from foreign animals such as mice and rats;
As a matter of course, a new problem has arisen in that administration into the human body can cause serious side effects such as anaphylaxis and other allergic reactions. In order to solve this problem, there are currently strong expectations that anti-cancer antibodies derived from humans rather than from xenobiotics will be developed and applied clinically.

On the other hand, there are currently many problems with the development of anti-human cancer human monoclonal antibodies. For example, artificial immune manipulation of cancer antigens cannot be performed unlike in xenogeneic animals, and above all, the stability of the resulting antibody-producing hybridomas is extremely poor.

Means for Solving the Problems Based on this technical background, the present inventors have made efforts to stably produce human MoAb that has the ability to bind to human solid tumor cells, and to apply it to the clinic. As a result of our research, we found that by using a mouse-human heterohybridoma of human lymphocytes and mouse myeloma cells as a parent strain, and by fusing the parent strain with lymphocytes derived from solid tumor patients, we could develop a long-term human MoAb that binds to human solid tumor cells. They obtained a hybridoma that stably produced over a long period of time, conducted further research, and completed the present invention.

That is, the present invention provides a (mouse/human)-human hybridoma that produces an anti-human solid tumor human monoclonal antibody,
The present invention relates to the antibody and the method for producing the antibody.

The anti-human solid tumor human monoclonal antibody of the present invention is useful as a therapeutic and diagnostic human immunoglobulin preparation, and can be used not only as an antibody alone but also as a substance with appropriate anti-human cancer activity, such as methotrexate, daunomycin, etc. It is combined with anticancer chemotherapeutic agents such as vincristine and cisplatin, or toxin proteins such as ricin, abrin, diphtheria toxin, and neocarzinostatin, as well as cytokines such as tumor necrosis factor, lymphotoxin, interferon, and interleukin, resulting in so-called missile therapy. It can also be used as an anti-cancer agent that specifically eliminates only cancer cells.

In addition, the anti-human solid tumor human monoclonal antibody-producing hybridoma of the present invention can be stably grown not only by culturing in a liquid medium but also by the asciticization method using nude mice, making it possible to prepare large quantities of antibodies, making it suitable for clinical application. It also has very excellent properties.

The present invention aims to produce anti-human solid tumor human monoclonal antibodies (
A mouse/human heterohybridoma was used as a parent strain to create a mouse/human)-human hybridoma. This mouse-human heterohybridoma is obtained by fusing human lymphocytes with mouse myeloma cells.

The human lymphocytes may be derived from the spleen, lymph nodes, peripheral blood, etc. of healthy individuals or patients with solid cancers, but peripheral blood lymphocytes of healthy individuals are particularly conveniently used. These human lymphocytes may be used for fusion as they are, but before fusion, they may be injected with B-cell stimulating factor 1, such as Hawkelyde mite-diene (hereinafter sometimes abbreviated as PWM), Staphylococcus Cowan IJ'C. It is preferable to stimulate and activate with mu serum and then subject to fusion.

Furthermore, as mouse myeloma cells, various known cells such as N5-1. P3UI, X45. Although Sp2 and the like are used, P2O3 is preferably used.

Cell fusion is performed using a known method, for example, polyethylene glycol (hereinafter sometimes abbreviated as PEG) is used as a fusion promoter to create a mouse/human heterohybridoma. It is desirable to make these mouse-human heterohybridomas HAT sensitive for selective culture during the next fusion, and for this reason, for example, 6-thioguanine (TG) or 8-azaguanine (AZ
G) to obtain these drug-resistant strains. Any of these HAT-sensitive mouse/human heterohybridomas can be used, but in particular, the known mouse/human heterohybridoma HM-5 [Ichimori et al., Biochemical and Biophysical Research Communications (BLochem, Biophy.
s, Res, Coml1. ), l 29 , 26
(1985), JP-A-61-12888] proliferates very stably and efficiently fuses with lymphocytes derived from solid tumor patients to obtain the (mouse-human)-human hybridoma of the present invention. Therefore, it is preferably used.

The (mouse/human)-human hybridoma of the present invention is created by fusing lymphocytes derived from a solid tumor patient with a mouse/human heterohybridoma and selectively culturing it in a medium supplemented with HAT.

Examples of the above-mentioned solid cancers include stomach cancer and colorectal cancer.

Esophageal cancer, liver cancer, pancreatic cancer, prostate cancer, lung cancer, ovarian cancer, uterine cancer.

Examples include breast cancer, bladder cancer, kidney cancer, and stomach cancer among others.

Colon cancer, liver cancer, and lung cancer are preferred.

Lymphocytes derived from solid cancer patients may be derived from spleen, lymph nodes, peripheral blood, etc., but lymph node lymphocytes are preferably used from the viewpoint of antibody-producing cell content and ease of acquisition. .

Although these human lymphocytes may be used for fusion as they are, it is preferable to stimulate and activate them with a B cell stimulatory factor such as pwM, Staphylococcus Cowan IJ mu serum, etc. before fusion, and then subject them to fusion.

Lymphocytes derived from a human solid tumor patient are incubated with a parent strain of mouse/human heterohybridoma in a medium containing a fusion promoter to undergo cell fusion. Examples of fusion promoters include PEG and Sendai virus, but in particular P
EG is preferably used. In the fusion method using PEG, the degree of polymerization of PEG is generally about t,ooo to 6,0
00. Incubation time is approximately 0.5-30 minutes, P
The concentration of EG used is about 10 to 80%. Preferably about 35-55% PEG 4,000 to 6,0
00 for about 1-10 minutes at about 37°C.

Various methods can be used to screen for hybridomas producing human monoclonal antibodies against human solid tumors.
For example, mixed hemagglutination reaction (hereinafter sometimes abbreviated as MHA), which involves adsorption of indicator red blood cells to cancer cells, and immunoadhesion reaction, which involves red blood cells adhering to antibodies bound to cancer cells via complement. (Hereinafter, it may be abbreviated as IA)
, fluorescently labeled second antibody (anti-human immunoglobulin antibody)
Immunofluorescence method (hereinafter referred to as IF) is a method of staining cancer cells with
(sometimes abbreviated as EL) or enzyme-linked immunosorbent assay (hereinafter referred to as EL)
cell-EL (sometimes abbreviated as ISA)
r SA method etc. Hybridomas that are positive for antibody activity are immediately subjected to cloning, which is usually easily accomplished by limiting dilution or the like. For culture supernatants of cloned (mouse/human)-human hybridomas, measure the antibody titer using the method described above, select hybridomas that stably produce high-titer antibodies, and prepare the desired monoclonal hybridoma. hybridomas can be obtained.

Generation of the anti-human solid tumor human monoclonal antibody.

Accumulation is usually carried out by culturing the (mouse/human)-human hybridoma of the present invention in a liquid medium or in the peritoneal cavity of a nude mouse, and examples of such culturing are given below.

As a liquid medium, for example, a basal medium for animal cell culture [e.g., a mixed medium of equal volumes of Iskot's medium and Ham's FI2 medium (■・
H medium), RPMI-1640 medium, etc.] supplemented with fetal bovine serum (hereinafter sometimes abbreviated as Fe2), or GIT medium (sold by Wako Pure Chemical Industries, Ltd.)
[See Japanese Unexamined Patent Publication No. 60-145088]. Cultivation is usually carried out for about 3 to 60 days, preferably for about 5 to IO
It is carried out at about 30-38°C, preferably about 37°C, for days.

For intraperitoneal transplantation into nude mice, [approx.
XlO'~5X107 antibody-producing hybridomas, preferably about 5x10', are transferred intraperitoneally, and about 10-35
A sufficient amount of antibody-containing ascites fluid can be obtained for 3 to 10 days after breeding for 1 day.

Antibodies in a liquid medium or ascites fluid can be purified using a combination of known biochemical techniques. For example, after centrifuging the antibody-containing solution, the supernatant is salted out (usually using ammonium sulfate or sodium sulfate), the resulting protein precipitate is dissolved in an appropriate buffer,
Post-dialysis column chromatography (ion exchange column,
The target antibody can be separated and purified by applying the antibody to a gel filtration column (gel filtration column, etc.). Separation as above,
By the purification operation, for example, about 5 to 15 g of anti-human solid tumor human MoAb with a purity of 90% or more in protein weight ratio can be obtained from a 500-ml liquid medium.

In addition, approximately 5 to 70 similar antibodies were found in the ascites fluid of 501R1.
B can be obtained. In these purified antibody preparations, the content of foreign proteins such as bovine serum albumin and bovine immunoglobulin or mouse-derived impure proteins is approximately 0.1% or less, so they are convenient when administered to humans as medicines. .

The human MoAb obtained as described above is treated with proteolytic enzymes (papain, pepsin, etc.), reducing agent treatment, etc. to obtain P that retains its ability to bind to human solid tumor cells.
Fragments of ab, Pab', F(ab') can be obtained and used for the same purpose as the human MoAb of the present invention.

The present invention specifically recognizes human solid tumor-specific antigens or human solid tumor-related antigens on the surface of human solid tumor cells, and binds to human solid tumor cells, but does not react with human fibroblast normal cells or human fetal tissues. Human solid cancer human monoclonal antibodies are produced, and among them, anti-human solid cancer human monoclonal antibodies exhibiting the following characteristics can be produced.

■Recognized antigen type: Cancer cell membrane surface type ■Reactivity with human cancer cells: Gastric cancer (NU(, C-4゜K)
ATO-Ill), colorectal cancer (SW-1222), lung cancer (
ADLC-DA).

■Reactivity with human normal cells: Fibroblast cells (LOVE
5kin, W I-38).

■Does not react with human fetal tissue (stomach, small intestine, lung, liver, kidney, thymus, tile).

The anti-human solid tumor human monoclonal antibody of the present invention exhibiting the above-mentioned properties can be used, for example, in clinical chemistry (C
1inical Chemistry), 27,
1797-1806 (1981), Journal of
Immunological Methods (J, 15Iluno1.
Methods).

80, tol-116 (1985), etc., and can be used as a diagnostic agent for human solid cancers in the diagnosis of healthy individuals and patients with solid cancers.

Since the anti-human solid tumor human monoclonal antibody of the present invention is homogeneous and has high titer, it is extremely suitable for use as a biochemical preparation. For example, after sterilization by filtration using a membrane filter or the like, it can be formulated as an injection or the like by itself or by mixing with an appropriate pharmacologically acceptable carrier, excipient, diluent, etc.

The human MoAb preparation obtained as described above can be used simply for analysis of human solid tumor-specific antigen or human solid tumor-related antigen.
It can be used not only for detection of blood antigens or as a diagnostic agent, but also for administration to humans as a prophylactic or therapeutic agent.

In particular, anti-human solid tumor human monoclonal antibodies as anti-cancer agents are expected not only to have anti-cancer effects themselves, but also to be administered to patients with solid tumors as carriers of appropriate anti-cancer agents, that is, as missile therapy agents, to eradicate solid tumors. There is.

As described above, since the anti-human solid tumor human monoclonal antibody of the present invention is derived from humans, it has extremely low antigenicity and toxicity for humans compared to antibodies derived from foreign animals such as mice and rats, and has almost no side effects. It can be administered directly to humans for prevention and treatment. Furthermore, the antibody-producing (mouse/human)-human hybridoma can stably produce antibodies in vitro with good yield, and therefore antibodies with high binding ability and constant quality can be stably produced in large quantities.

Hereinafter, the present invention will be explained in more detail with reference to Reference Examples and Examples, but the present invention is not limited thereto.

The (mouse/human)-human hybridoma 71-5.2F9 disclosed in Example 3 was deposited with the Fermentation Research Institute (IFO) on March 2, 1985 under the accession number rpo.
! ;o/63, and has been deposited with the Microbial Research Institute (FRI) of the Ministry of International Trade and Industry since March 23, 1985, under the accession number FERM Hp-/go.

Reference example 1 Mixed hemocytosis assay (MHA) Among the target cells, adherent cells are 24 or 4
8 hours ago, the 60-well microplate (NUN
C) and cultured at 500 cells/well. For floating cells, suspend them in serum-free culture medium on the day of the test, dispense the same number of cells into each well, and incubate at 400Xg.
, centrifuged for 5 minutes and attached to the plate.

To prepare indicator blood cells, Rh+ human type 0 blood cells were washed three times with phosphate saline buffer (PBS) and made into a 2% suspension in PBS, and this suspension was diluted 16 times with PBS to determine human blood type. Equal amounts of anti-Rh antibody (manufactured by Ortho) were mixed and reacted at 37°C for 30 minutes. PB this blood cell
Washed three times with S and resuspended at 2%. Next, 25 on PBS
Rabbit anti-human IgG antibody (DAK
(manufactured by O) Co., Ltd.) were mixed in equal amounts and further reacted at 37° C. for 130 minutes. Thereafter, it was washed three times with PBS and stored as a 2% suspension.

The plate with attached cells was treated with 0.1M MgCQ,
-0,03M CaCC, -0,1% glucose diethylbarbitate saline buffer (veronalbuf
fered 5aline), pH 7,4 (V B
After washing with a solution containing 5% fetal bovine serum (Fe2) in M), the culture supernatant or ascites was dispensed into each well and allowed to stand at room temperature for 1 hour. After washing the plate with VBM, 5% FO
Indicator blood cells diluted to 0.2% with 9-VBM were dispensed into each well and allowed to stand at room temperature for 40 minutes. Next, remove the unreacted blood cells by VB
It was removed by washing with M and observed under a microscope. A positive reaction was defined as a case where rosette formation was 1% or less in the reaction negative control well to which no antibody was added, and rosette formation was observed in 25% or more of the target cells in the wells of this test.

Reference Example 2 Immunoadsorption (IA) Preparation of target cells and observation and judgment of results were performed in the same manner as the MHA method. The antibody reaction was carried out by standing in a humid chamber at 4°C for 1 hour. Add P to the plate washed with VBM.
A mixture of 0.2% human type O red blood cells washed with BS and 1/60 to 1/100 volume of fresh guinea pig serum was added to VBM, and the mixture was dispensed into each well and incubated at 37°C for 30 minutes with 5% CO1 added. It was left standing in an aerated incubator. Next, unreacted blood cells were washed away with VBM, and the results were observed and judged.

Reference Example 3 Immunofluorescence method (IP) Frozen sections were prepared from human tumors, fetal tissues, etc. transplanted into nude mice, which were quickly frozen in OCT compound (Miles) and stored at -80°C, and fixed with acetone. and subjected to fluorescent antibody staining.

First, culture supernatant or appropriately diluted ascites was placed on the section and allowed to react at 37° C. for 40 minutes in a humid chamber. After washing three times for 10 minutes with PBS, it was washed once with distilled water and dried.

Next, FITC-labeled anti-human IgM appropriately diluted with PBS
It was reacted with an antibody (manufactured by TAGO) in the same manner, and washed twice with PBS for 10 minutes. Additionally, 200 PBS and 2% Evans Blue were added.
Counterstain (c) in a solution with a few drops of
5 stain) was performed. This staining solution was washed with distilled water, dried, and washed with 50% glycerin-PBS.
After mounting, the samples were observed using a fluorescence microscope. The results were determined using the culture supernatant of mouse-human heterohybridoma HM-5 or the serum of untreated nude mice as a negative control.

Example 1 Establishment of 8-azaguanine-resistant strain of mouse/human heterohybridoma (i) Production of mouse/human heterohybridoma Lymphocytes (PBL) were extracted from terminal blood of a healthy individual by specific gravity centrifugation using Ficoll-Hypaque. Separate, IO%
r-H medium containing immobilized F'C9 (■・I-■-1
0F) so that 2×10' pieces/- were floated.

This lymphocyte suspension was dispensed into a tissue culture flask (Corning F'-150 flask), and 3.2 μg/-
PWM was added to the cells so that the microscopicity of the cells was reached, and the cells were cultured at 37° C. in a carbon dioxide gas atmosphere for 3 days.

After culture, lymphocytes and HGPRT-deficient HAT-sensitive mouse myeloma cells P3-x63-Ag8. Ul(P3UI)
(Current Topics in Microbiology and Immunology (Cur, Topics.

Microbiol, Is+iun, ), 8 above, 1-
7 (1978)) at a cell ratio of 2:1, and 7
Cell fusion is performed by treating for 1 minute. After fusion, tumor cells!・The cells were suspended in H-10F at a concentration of 2xlO'/cell, seeded 1 d each in a Linpro 24-well multi-dish, and cultured at 37°C in a carbon dioxide gas atmosphere. Cultivate! After 124 hours, HAT (hyboxanthin: 1X
10''''4M, aminopterin: 4 x 10''-'M, thymidine 1.6 x 10-'M)
) (AT medium) was added to start HAT selection culture. In addition, HAT selective culture was continued by picking up the limbs and adding 1-1 fresh HAT medium 3,5,7 odd days after the day of initial addition. Hybridoma growth was observed 10-14 days after cell fusion.

(ii) Obtaining an 8-Azaguanine-resistant strain In order to make the mouse-human heterohybridoma obtained in (i) with excellent growth ability and no antibody production into a HAT-sensitive strain, an 8-AZG-supplemented medium was added. Culture was carried out. That is, RPMI-1 containing lμMa degree of 8-AZG
Cultivation was started at 0F, and the 8-AZGa concentration was increased sequentially by 2 times from 18M, and cultivation was continued. 100 μM 8-AZ
For strains that have become resistant to G, examine HATg susceptibility.
Susceptible mouse-human heterohybridoma I (M-5
() (M'-5 strain) was obtained.

Example 2 (Mouse/Human) - Creation of human hybridoma A lymph node from a gastric cancer patient was cut into small pieces with small scissors, and a lymph cell suspension was collected by passing it through a stainless mesh and washed with RPMI-1640 medium. . The obtained cell suspension (2XlO” cells/rn1.) was subjected to PWM (
Along with a 100-fold diluted solution of GIBCO (manufactured by GIBCO),
After culturing and activating for 4 days, the cells were subjected to cell fusion.

Cell fusion was carried out by mixing the above activated human lymphocytes and the HM-5 strain obtained in Example 1 at a ratio of 5:1, and further adding 42.5% PEG as a fusion promoter. 37
°C, mix for 1 minute, after fusion,! Summer IAT including 0%FCS
Selective culture was started in a medium containing RPMI-1640. The medium was replaced with fresh medium as appropriate, and hybridoma growth was observed 10 to 14 days after cell fusion.

Anti-human solid tumor antibodies in the culture supernatant were measured and screened using the MHA and IA methods described in Reference Examples 1 and 2, and the production of antibodies capable of binding to human solid tumor cells (mouse and
Human)-human hybridomas were collected (Table 1).

Table 1 Experiments to obtain hybridomas producing anti-human cancer antibodies
Seeding Seeding Growth Antibody positive well 2) ■) 1 1XIO' 768 768
32 222 1x105 368 3
68 6 13 5xlO'
384 384 0 14
1XlO' 480 451 1
05 2.5xlO' 88 88
'O165xlO' 1248 3
98 0 075xlO' 7
52 556 11 91) Number of activated lymphocytes seeded per t-well.

2) Reference example! The search was conducted using the methods described in , and 2.

Example 3 Cloning of hybridomas producing anti-human solid tumor human monoclonal antibodies Among the hybridomas producing anti-human solid tumor human monoclonal antibodies obtained in Example 2, one with a high antibody titer was selected.
Cloning was performed by the limiting dilution method. That is,
The hybridomas were suspended in RPMI-1640 medium supplemented with 10% FC9 at a concentration of 10°30 and 90 cells/rn1, and dispensed into a 96-well microplate at 100 μQ/well each. BALB as a feeder cell
/c Mouse thymocytes were added at 2XIO' per well. In this way, cell proliferation was observed after about 2 weeks, and when the antibody titer of the culture supernatant was measured and screened using the MHA and IA methods shown in Reference Examples 1 and 2, some clones were found to be solid. Antibody activity binding to cancer cells was observed. Among them, the (mouse/human)-human hybridoma 71-5.2F9, which has particularly excellent proliferation ability and high antibody production ability, was selected and bred (FE
RM BP-7 and 08. [FOTA o/63).

Example 4 Production of human monoclonal antibodies (mouse and
human)-human hybridoma 71-5゜2F9 (FER
After acclimating M Bp-1Ho&, IFOdaO/63) to GET medium, the same G
The cells were suspended in ET medium and cultured at 37°C for about 5 days. Ammonium sulfate was added to 500 d of the obtained culture supernatant to a concentration of 45% and salted out at 4° C. for 60 minutes. The obtained protein precipitate was dissolved in 2hM phosphate buffer (pH 7,9) containing 0.2M NaCσ, and dialyzed against 3 g of the same buffer. After exchanging the external dialysis solution twice, it was subjected to high performance liquid chromatography using a TSK gel G3000SW gel filtration column (manufactured by Toyo Soda) conditioned with the same phosphate saline buffer. By collecting and concentrating the flow-through fractions with a molecular weight of 500,000 or more, anti-human solid tumor human MoAb71-5
．． Approximately 9 fg of 2F9 was collected (Table 2).

Table 2 Anti-human solid tumor human monoclonal antibody 71-5
．． 2P9 purified human bovine bovine serum (ml (mg) (mg) (mg)
(mg) Culture supernatant 500 1,65fj13
4.6 680 ammonium sulfate salting out 7 28 1
0 0.029 0.042 45% ammonium sulfate 2) High performance liquid chromatography using TSK gel G3000SW column Example 5 Production of human monoclonal antibody ■ (mouse/human) - Human hybridoma 71-
5゜2F9(FERM BP-/?0♂, I F"O
0763) was cultured in RPMI-1640 medium supplemented with 10% FCS, washed with physiological saline, and suspended at a concentration of 5×10 8 cells/d. Then 1M1. The above hybridoma suspension was intraperitoneally transplanted into nude mice that had been previously intraperitoneally inoculated with Bristan (manufactured by Aldrich Chemical Co.), and ascites fluid was collected 3 to 5 weeks later. The ascites fluid was diluted with phosphate saline buffer and then subjected to ammonium sulfate salting out and high performance liquid chromatography in the same manner as the culture supernatant in Example 4. As a result, approximately 35 mg of human M
oAb71-5.2F9 was obtained.

In addition, binding of the antibody to target cancer cells was performed using IA described in Reference Example 2.
The antibody dilution curve of ascites fluid was determined by the method. The results were as shown in Figure 1.

In FIG. 1, the vertical axis is human MoAb71-5, 2
The percentage (%) of cells bound to F9 is shown, and the horizontal axis shows the ascites dilution factor. α---Li 1 bite---One bite.

△---△ indicates that the target cancer cells are NUGC-4°HC.
T-15,5W-480, -1 indicates that the target cancer cells are 5K-Co-1,5W-1083, Cap
Indicates that it is an-1 or BXPC3.

Example 6 Characteristics of human monoclonal antibody 71-5.2F9 Human MoAb 71-5.2 obtained in Examples 4 and 5
The properties of F9 were determined by the IA method and I method shown in Reference Examples 2 and 3.
When measured using the F method, the results shown in Tables 3 and 4 were obtained.

Table 3 shows the reactivity against various fetal tissues and human cancer cells that can be transplanted into nude mice, as measured by the IF method. The antibody of the present invention did not react with fetal tissue at all, but specifically reacted only with the cancer cells of the rosary.

Table 4 shows the reactivity against various cancer cells and normal cells measured by the IA method.

It did not react with normal cells but showed specific binding ability to several types of cancer cells.

The immunoglobulin class of the antibody of the present invention was determined to be human 1gM type by Ophthalony method.

Furthermore, in Table 5, the properties of cell surface antigens recognized by the antibodies of the present invention were examined. Target gastric cancer cell NUGC-
Heat treatment, trypsin treatment, or neuraminidase treatment of 4 did not change the antibody binding ability to cells, whereas periodic acid treatment caused loss of antigenicity of cells.
Human MoAb71-5.2F9 of the present invention was found to recognize cancer cell membrane surface type, asialo type sugar chain related antigen.

Table 3 Reactivity of human monoclonal antibody 71-5.2F9 to target cells and NU to target cells and tissues
GC-4 Gastric cancer 10PTGC-1- PTGC-2+ Co-3 Colon cancer - 1uc i~3 Lung cancer Ca1
u-1- L-27- SCLC-MO- AM-OV-1 melanoma - Stomach Fetal tissue - Small intestine - Lung
-Liver
-kidney
-thymus gland
- Shin - Table 4 Specificity of human monoclonal antibody 71-5.2F9 2) Positive cells: Gastric cancer (NUGC-3, -4, KAT
O-II [, MKN-28).

Colorectal cancer (SW-403, -480, -1222, 5K-
Co-17°HCT-15), lung cancer (ADLC-DA,
MH), sarcoma-negative cells: Cancer cell line [gastric cancer (NU
GC-2, MKN-1, AM-GC-1).

Colorectal cancer (SW-620, -1083, -1116, -1
417,5K-CO-1,-15,HCT-116,I
T-29, LOVO, CaCo-2).

Lung cancer (SCLC-12, -13, -17, -MO, Lu
ci-4, -6゜SK-MES-1, MM, MT, OT
, SA, SM, MOA, Ca1u-1゜5BC-3,0
G-90, PC-10, Lu-134, A-431,8
57).

Pancreatic cancer (Capan-1, -2, BxPc-3, AsPc
-1), liver cancer (HepG-2), renal cancer (SK-RC-1)
8, Dematoley.

5cato1a, Yiola, Nometh), bladder cancer (T-24, KK-47), breast cancer (BT-20),
Cervical cancer (ME-180), ovarian cancer (SK-OV-3,
CoLo-3, ROAC, ARA, Kl).

Melanoma (SK-MEL-28, -33, -37, M
-14).

Neuroblastoma (SK-N-8H, GOTO, CHP), glioma (SK-MG-1, Becker).

Sarcoma (U-20-S), T-cell leukemia (HUT)
-78, -102, MT-1, -2jURKAT, MO
LT-3, SOMMER.

CCRF-CEM, HPB-ALL), 13-cell leukemia (Raj i.

Daudl), myeloid leukemia (U-937), null cell normal cell line [fibroblast (LOVE 5kin, Wb2
) Measured by the IA method described in Reference Example 2.

Table 5 Characteristics of cell membrane antigen recognized by human monoclonal antibody 71-5.2F9 Target cell treatment method Antibody binding ability to target cell 1) 1oo=. , to minutes. Heat treatment +2)3
7. , 30 minutes. . -25% + 2
) Trypsin digestion treatment at room temperature, 2% of tomin 3) NalOi
Oxidation treatment at 37°C, 30 minutes OIU/, +3) l) Gastric cancer cell line NUGC-4 was used.

2) Measured by the IA method described in Reference Example 2.

3) Measured by the IF method described in Reference Example 3.

[Brief explanation of the drawing]

FIG. 1 shows an antibody dilution curve of the ascites fluid of a nude mouse transplanted with the (mouse/human)-human hybridoma 71-5.2F9 obtained in Example 5.

Claims (10)

[Claims] (1) Produce an anti-human solid tumor human monoclonal antibody obtained by fusing a mouse-human heterohybridoma obtained by fusing human lymphocytes and mouse myeloma cells with lymphocytes derived from solid tumor patients (mouse・Human) - human hybridoma. (2) The hybridoma according to claim 1, wherein the mouse myeloma cell is P3U1. (3) The hybridoma according to claim 1, wherein the mouse-human heterohybridoma is mouse-human heterohybridoma HM-5. (4) The hybridoma according to claim 1, wherein the lymphocytes derived from a solid cancer patient are lymphocytes derived from a lymph node of a solid cancer patient. (5) (Mouse/human)-human hybridoma 71-5.2F obtained by fusion of mouse/human heterohybridoma HM-5 and lymphocytes derived from lymph nodes of gastric cancer patients
9. (6) An anti-human solid tumor human monoclonal antibody that does not react with normal human fibroblast cells or human fetal tissues and binds to human solid tumor cells. (7) The antibody according to claim 6, which has the following properties. [1] Type of antigen recognized: Cancer cell membrane surface type [2] Reactivity with human cancer cells: Gastric cancer (NUGC-4, K
ATO-III), colorectal cancer (SW-1222), lung cancer (A
DLC-DA) positive [3] Reactivity with human normal cells: fibroblasts (LOVE
skin, WI-38). [4] Does not react with human fetal tissues (stomach, small intestine, lung, liver, kidney, thymus, spleen). (8) Anti-human solid tumor human monoclonal antibody 71-5.
2F9. (9) A method for producing an anti-human solid tumor human monoclonal antibody, which comprises culturing the hybridoma according to claim 1 in a liquid medium or intraperitoneally of a nude mouse. (10) The production method according to claim 9, wherein the hybridoma to be cultured is the hybridoma according to claim 5.